WO1994028925A1 - Vaccine containing a serine protease - Google Patents

Vaccine containing a serine protease Download PDF

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Publication number
WO1994028925A1
WO1994028925A1 PCT/GB1994/001274 GB9401274W WO9428925A1 WO 1994028925 A1 WO1994028925 A1 WO 1994028925A1 GB 9401274 W GB9401274 W GB 9401274W WO 9428925 A1 WO9428925 A1 WO 9428925A1
Authority
WO
WIPO (PCT)
Prior art keywords
vaccine
dipeptidyl peptidase
amc
protease
activity
Prior art date
Application number
PCT/GB1994/001274
Other languages
English (en)
French (fr)
Inventor
John Pius Dalton
Stuart J. Andrews
Original Assignee
Mallinckrodt Veterinary Inc.
Matthews, Derek, Peter
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mallinckrodt Veterinary Inc., Matthews, Derek, Peter filed Critical Mallinckrodt Veterinary Inc.
Priority to SI9430241T priority Critical patent/SI0703789T1/xx
Priority to NZ267139A priority patent/NZ267139A/en
Priority to DK94917738T priority patent/DK0703789T3/da
Priority to JP7501508A priority patent/JPH08511262A/ja
Priority to AU69333/94A priority patent/AU694121B2/en
Priority to DE69416405T priority patent/DE69416405T2/de
Priority to US08/564,091 priority patent/US5885814A/en
Priority to EP94917738A priority patent/EP0703789B1/de
Publication of WO1994028925A1 publication Critical patent/WO1994028925A1/en
Priority to GR990401198T priority patent/GR3030116T3/el

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to the use of a class of serine proteases as protective antigens against helminth parasites,
  • Each species of domestic animal can be parasitised by a number of different species of helminths, a process which usually causes disease.
  • the parasitic trematode Fasciola hepatica is known to-be the cause of the economically important disease fascioliasis in ruminants, such as cattle and sheep.
  • the parasite enters the mammalian host by penetrating the gut wall and spends approximately seven weeks feeding on and burrowing through the liver mass before migrating into the bile duct. Following infection, development of immunity in the host can be poor and resistance to reinfection in already infected hosts may be only partial or non-existent.
  • Other parasitic flukes include Fasciola ⁇ i ⁇ antica and Dicrocoelium spp. and also Paramphistomum spp.
  • nematodes such as hookworms (e.g. Necator. Ancylostoma, Uncinaria and Bunostomum sp . ) .
  • parasitic worms of economic importance include the various species of the following helminth genera:- Trichostron ⁇ ylus. Nematodirus. Dictyocaulus. Cooperia. Ascaris. Dirofilaria, Trichuris and Stron ⁇ ylus .
  • helminth genera include the various species of the following helminth genera:- Trichostron ⁇ ylus. Nematodirus. Dictyocaulus. Cooperia. Ascaris. Dirofilaria, Trichuris and Stron ⁇ ylus .
  • pets and humans may also be infected, not infrequently with fatal results and helminth infections and infestations thus pose a problem of considerable worldwide significance.
  • Control of helminth parasites of grazing livestock currently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques are often unsatisfactory, firstly because anthelmintic drugs may have to be administered frequently, secondly because resistance against anthelmintic drugs is - becoming increasingly widespread and thirdly because appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
  • a vaccine against F.hepatica has been proposed in W090/08819 comprising a glutathione-S-transferase from F.hepatica as antigenic material.
  • Bennett extracted various antigens from Fasciola organisms by a process which separates antigens specific to the juvenile stage from antigens present throughout the juvenile and adult stages.
  • a first aspect of the present invention provides a vaccine for use in combating a parasitic infestation of helminths in a mammal wherein the antigenic material comprises a serine protease having dipeptidyl peptidase-like activity, in at least partially purified form, or an antigenic fragment or epitope thereof, together with a carrier and/or adjuvant.
  • the invention also provides a method of combating a parasitic infestation of helminths in a mammal comprising administering to said mammal a vaccine according to the invention as hereinbefore defined in an amount effective to combat said infestation.
  • the mammal is preferably a ruminant, for example cattle or sheep, but the vaccine and method of the invention may also find application in humans.
  • the dipeptidyl peptidase-like protease is derived from flukes such as Fasciola or Dicrocoelium. in particular from the liver fluke Fasciola hepatica.
  • the dipeptidyl peptidase should be capable of stimulating an immune response which will be effective against Fasciola or Dicrocoelium. in particular F. hepatica and F. ⁇ i ⁇ antica. such dipeptidyl peptidase-like proteases from other species as are capable of conferring a cross- protective immune response thus forming a particularly preferred aspect of the invention.
  • the F.hepatica dipeptidyl peptidase-like protease shown hereinafter to possess a molecular weight of approximately 200 KDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing and non-reducing conditions and by gel filtration chromatography is particularly preferred for use in the vaccine and method of the invention and as a novel protein itself forms a further aspect of the invention.
  • the dipeptidyl peptidase incorporated in the vaccine according to the invention is in at least partially purified form.
  • the dipeptidyl peptidase comprises at least 75% of the total excretory/secretory proteins present in the vaccine and more preferably the dipeptidyl peptidase is at least 95% pure. It will be appreciated that once dipeptidyl peptidase of at least 95% purity has been obtained it can be admixed with one or more further purified antigenic proteins, including one or more further excretory/secretory proteins, to form a polyvalent vaccine.
  • a preferred form of polyvalent vaccine according to the invention will contain a dipeptidyl peptidase as referred to above in combination with a Cathepsin L-type antigen as described in more detail in International Patent Application No. WO94/09142.
  • Such a polyvalent vaccine will, by inducing immunity in the host species against two separate aspects of the invading helminth parasite, significantly increase the likelihood of protection against the helminth and significantly reduce the chances of infestation occurring.
  • Dipeptidyl peptidases have been isolated from a mammalian source (J. Biol. Chem. 263 (1988) , pages 6613- 6618) and were characterised by an ability to cleave dipeptide substrates.
  • dipeptidyl peptidases of the present invention are likewise characterised by their ability to cleave fluorogen linked dipeptide substrates while showing no activity against fluorogen linked mono amino acid substrates thus demonstrating that the enzymes cleave amino acids in pairs from the N-terminus of the substrate.
  • the vaccines according to the invention may be formulated with conventional carriers and/or adjuvants and the invention also provides a process for the preparation of the vaccines comprising bringing into association purified dipeptidyl peptidase or an antigenic fragment or epitope thereof and one or more adjuvants or carriers.
  • Suitable adjuvants include aluminium hydroxide, saponin (ISCOMs) , quil A and more purified forms thereof, muramyl dipeptide, mineral and vegetable oils, DEAE dextran, nonionic block copolymers or liposomes such as Novasomes (Trade Mark of Micro Vesicular Systems Inc.) , in the presence of one or more pharmaceutically acceptable carriers or diluents.
  • Carriers for peptide sequences corresponding to epitopes of dipeptidyl peptidases according to the invention can be proteins such as Hepatitis B core antigen multiple antigen peptide or lipopeptides such as tripalmitoyl-S- glycerylcysteinylserylserine (P 3 CSS) .
  • Suitable diluents include liquid media such as saline solution appropriate for use as vehicles. Additional components such as preservatives may be included.
  • Administration of the vaccine to the host species may be achieved by any of the conventional routes, e.g. orally or parenterally such as by intramuscular injection, optionally at intervals e.g. two injections at a 7-35 day interval .
  • a suitable dose when administered by injection might be such as to give an amount of dipeptidyl peptidase protein within the range 10-500 ⁇ g.
  • the dipeptidyl peptidase for use in the vaccine according to the invention may be prepared by isolation from the excretory/secretory products of adult and/or juvenile helminths, it may also be convenient to prepare it by recombinant DNA techniques with the known advantages which such techniques give in terms of scaling-up of production and reproducibility.
  • the invention also provides a dipeptidyl peptidase or a proenzyme therefor or an antigenic fragment or epitope thereof, produced by means of recombinant DNA techniques.
  • Additional aspects of the invention related to the above include DNA molecules encoding for dipeptidyl peptidases or antigenic fragments or epitopes thereof; vectors containing one or more such DNA sequences; host cells, for example bacteria such as E. coli or more preferably eukaryotic cells, transformed by such vectors, for example by a baculovirus vector; and processes for preparing recombinant dipeptidyl peptidase or antigenic fragments or epitopes thereof comprising culturing such transformed host cells and isolating said dipeptidyl peptidase or fragment or epitope from the cultured cells.
  • An alternative live or inactivated vaccine formulation may comprise an attenuated or virulent virus or a host cell, e.g. a microorganism such as a bacterium, having inserted therein a nucleic acid molecule (e.g. a DNA molecule) according to the invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
  • a bacterial vector which elicits local gut mucosal immunity to a fluke antigen which then blocks juvenile fluke migration is particularly preferred, notably invasive species such as Salmonella species.
  • Additional antigenic materials may also be present in the vaccine thus giving an enhanced protective effect against the helminth parasite in question or a combined protective effect against one or more additional parasitic infestations.
  • a yet further aspect of the invention provides a monoclonal or polyclonal antibody capable of inducing immunity to a dipeptidyl peptidase in a mammal when administered to said mammal, the antibody having an affinity for the variable region of one or more further antibodies, said further antibodies having an affinity for said dipeptidyl peptidase.
  • Mature Fasciola hepatica flukes were removed from the bile ducts of bovine livers obtained at an abattoir in Ireland. The flukes were washed six times in phosphate buffered saline (PBS) , pH 7.3, and then maintained in Roswell Park Memorial Institute (RPMI) - 1640, pH 7.3, containing 2% glucose, 30 mM Hepes and 25 mg/1 gentamycin overnight at 37°C. The medium (excretory/secretory or E/S products) was then removed, frozen and stored at -20°C.
  • PBS phosphate buffered saline
  • RPMI Roswell Park Memorial Institute
  • the activity of the protease on both gly-pro-AMC and lys-ala-AMC substrates was estimated over the pH range 4.5 to 8.2.
  • the pH for optimum activity was determined to be pH 6.8. It was also demonstrated that the activity of the protease was optimum in HEPES buffer,- both tris and phosphate buffers adversely affected the activity (see Table 1) .
  • HEPES buffer 50 mM pH 6.8) has therefore been the routine buffer used in all experiments and purification procedures.
  • Peptide bond specificity was investigated using several fluorogenic peptide substrates that have previously been used to categorise mammalian dipeptidyl peptidases I, II, III and IV (J. Biol Chem. 263 (1989) , pages 6613- 6618) . These substrates included gly-arg-AMC (DPP-I) , lys-ala-AMC (DPP-II) , arg-arg-AMC (DPP-III) and gly-pro- AMC (DPP-IV) .
  • DPP-I gly-arg-AMC
  • DPP-III lys-ala-AMC
  • DPP-III arg-arg-AMC
  • DPP-IV gly-pro- AMC
  • the assay mixture contained 200 ⁇ l of 20 ⁇ M substrate, 100 ⁇ l of enzyme solution and 900 ⁇ l of 50 mM Hepes, pH 6.8.
  • the reaction was allowed to proceed at 37°C and stopped by the addition of 200 ⁇ l of 1.7M acetic acid.
  • the liberated AMC was measured in a fluorometer with excitation and emission wavelengths of 370 nm and 440 nm, respectively,
  • the liver fluke protease selectively cleaved lys-ala-AMC and gly-pro-AMC, the Michealis-Menton constants (K for these substrates being 58 and 25 ⁇ M, respectively.
  • the protease did not cleave the substrates gly-arg-AMC and arg-arg-AMC.
  • K m affinities for these two substrates
  • the fluke enzyme shows both DPP-II and DPP-IV type activity if the classification applied to mammalian DPPs is used.
  • the enzyme showed no activity against various aminopeptidase substrates including lys-, ala-, leu- and pro-AMC.
  • samples of E/S were electrophoretically separated in 10% native polyacrylamide gels (prepared as described above with the exception that no SDS was added to samples or gels) and following electrophoresis the gels were immersed in solutions of the various fluorogen linked substrates (50 ⁇ M in 50 mM HEPES) . After incubation the gels were then placed on a UV transilluminator and photographed using polaroid film. Only a single band was visualised, with an identical mobility, using the substrates gly-pro-AMC and lys-ala- AMC.
  • the effect of various metal ions on the hydrolytic activity of the enzyme was examined using both gly-pro- AMC and lys-ala-AMC as substrates in assays as described above.
  • the results demonstrate that metal ions such as calcium, magnesium and manganese have little effect on enzyme activity whereas zinc at 1 mM concentration inhibits activity by >85%.
  • the heavy metals iron, mercury, cadmium and cobalt all inhibit enzyme activity to >90% at a concentration of 5mM whereas lead showed ⁇ 20% inhibition at this concentration.
  • the enzyme was not inhibited by the proteinase inhibitors EDTA, pepstatin and aprotinin indicating that the enzyme is not a metallo- or aspartyl protease.
  • the enzyme is inhibited by phenylmethyl sulfonyl fluoride (PMSF) , an inhibitor of serine and cysteinyl proteinases; however, since no activation of the enzyme is observed with thiol activating agents such as cysteine, dithiothreitol and mercaptoethanol the enzyme does not appear to be a cysteinyl proteinase.
  • PMSF phenylmethyl sulfonyl fluoride
  • Benzamidine an inhibitor of serine proteinases, inhibits the activity of the DPP.
  • liver fluke enzyme is a serine proteinase. These inhibition studies also support our conclusion that a single enzyme cleaves both gly-pro-AMC and lys-ala-AMC substrates as a similar inhibitor profile was observed when either substrate is used (Table 3) .
  • Mammalian DPP2 and DPP4 can be distinguished using the inhibitors puromycin and bacitracin; puromycin will inhibit DPP2 only whilst bacitracin inhibits DPP4 only (J. Biol. Chem. 2£1 (1988), 6613-6618) .
  • puromycin will inhibit DPP2 only whilst bacitracin inhibits DPP4 only (J. Biol. Chem. 2£1 (1988), 6613-6618) .
  • both these substrates were tested in inhibition studies with the fluke enzyme neither showed any inhibitory effect highlighting another difference between mammalian DPPs and the fluke enzyme (see Table 3 above) .

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PCT/GB1994/001274 1993-06-15 1994-06-14 Vaccine containing a serine protease WO1994028925A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
SI9430241T SI0703789T1 (en) 1993-06-15 1994-06-14 Vaccine containing a serine protease
NZ267139A NZ267139A (en) 1993-06-15 1994-06-14 Vaccine against helminths having a serine protease as antigen
DK94917738T DK0703789T3 (da) 1993-06-15 1994-06-14 Vaccine indeholdende en serinprotease
JP7501508A JPH08511262A (ja) 1993-06-15 1994-06-14 セリンプロテアーゼを含むワクチン
AU69333/94A AU694121B2 (en) 1993-06-15 1994-06-14 Vaccine containing a serine protease
DE69416405T DE69416405T2 (de) 1993-06-15 1994-06-14 Serinprotease enthaltender impfstoff
US08/564,091 US5885814A (en) 1993-06-15 1994-06-14 Vaccine containing a serine protease
EP94917738A EP0703789B1 (de) 1993-06-15 1994-06-14 Serinprotease enthaltender impfstoff
GR990401198T GR3030116T3 (en) 1993-06-15 1999-04-30 Vaccine containing a serine protease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB939312324A GB9312324D0 (en) 1993-06-15 1993-06-15 Vaccine
GB9312324.8 1993-06-15

Publications (1)

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WO1994028925A1 true WO1994028925A1 (en) 1994-12-22

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ID=10737190

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PCT/GB1994/001274 WO1994028925A1 (en) 1993-06-15 1994-06-14 Vaccine containing a serine protease

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US (1) US5885814A (de)
EP (1) EP0703789B1 (de)
JP (1) JPH08511262A (de)
AT (1) ATE176402T1 (de)
AU (1) AU694121B2 (de)
DE (1) DE69416405T2 (de)
DK (1) DK0703789T3 (de)
ES (1) ES2130426T3 (de)
GB (1) GB9312324D0 (de)
GR (1) GR3030116T3 (de)
HU (1) HUT71209A (de)
NZ (1) NZ267139A (de)
WO (1) WO1994028925A1 (de)
ZA (1) ZA944241B (de)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047740A2 (en) * 1996-06-11 1997-12-18 Mallinckrodt Veterinary Inc VACCINE CONTAINING A PEROXIREDOXIN AND/OR A β-TUBULIN
GB2327346A (en) * 1997-07-04 1999-01-27 Univ Melbourne Parasite vaccine
WO2004050883A2 (en) * 2002-12-04 2004-06-17 Institute Of Bioorganic Chemistry Chimaeric protein containing cysteine protease of liver fluke fused to hepatitis b core protein or ubiquitin, plants expressing said protein, and uses thereof as vaccine
WO2011043962A2 (en) 2009-10-07 2011-04-14 Wyeth Llc Compositions comprising adjuvant, macrolide and proteinaceous antigen and methods of use thereof
US20190145981A1 (en) * 2016-04-21 2019-05-16 Sphingotec Therapeutics Gmbh Methods for determining dpp3 and therapeutic methods

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030065382A1 (en) * 2001-10-02 2003-04-03 Fischell Robert E. Means and method for the treatment of coronary artery obstructions
EP1745292A2 (de) 2004-04-28 2007-01-24 Bayer HealthCare AG Diagnositka und therapeutika für mit dipeptidyl-peptidase 1 (dpp1) assoziierten krankheiten

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001277A1 (en) * 1986-08-18 1988-02-25 The Australian National University Helminth parasite vaccine
WO1994009142A1 (en) * 1992-10-21 1994-04-28 Mallinckrodt Veterinary, Inc. Vaccine containing a thiol protease

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879213A (en) * 1986-12-05 1989-11-07 Scripps Clinic And Research Foundation Synthetic polypeptides and antibodies related to Epstein-Barr virus early antigen-diffuse
NZ230773A (en) * 1988-09-26 1993-04-28 Biotech Australia Pty Ltd Nematode antigenic peptides and vaccines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001277A1 (en) * 1986-08-18 1988-02-25 The Australian National University Helminth parasite vaccine
WO1994009142A1 (en) * 1992-10-21 1994-04-28 Mallinckrodt Veterinary, Inc. Vaccine containing a thiol protease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Columbus, Ohio, US; *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047740A2 (en) * 1996-06-11 1997-12-18 Mallinckrodt Veterinary Inc VACCINE CONTAINING A PEROXIREDOXIN AND/OR A β-TUBULIN
WO1997047740A3 (en) * 1996-06-11 1998-03-26 Mallinckrodt Veterinary Inc VACCINE CONTAINING A PEROXIREDOXIN AND/OR A β-TUBULIN
US6676944B2 (en) 1996-06-11 2004-01-13 John P. Dalton Vaccine containing a peroxiredoxin and/or a β-tubulin
GB2327346A (en) * 1997-07-04 1999-01-27 Univ Melbourne Parasite vaccine
WO2004050883A2 (en) * 2002-12-04 2004-06-17 Institute Of Bioorganic Chemistry Chimaeric protein containing cysteine protease of liver fluke fused to hepatitis b core protein or ubiquitin, plants expressing said protein, and uses thereof as vaccine
WO2004050883A3 (en) * 2002-12-04 2004-11-04 Inst Of Bioorg Chemistry Chimaeric protein containing cysteine protease of liver fluke fused to hepatitis b core protein or ubiquitin, plants expressing said protein, and uses thereof as vaccine
AU2003295293B2 (en) * 2002-12-04 2009-12-03 Institute Of Bioorganic Chemistry Chimaeric protein containing cysteine protease of liver fluke fused to hepatitis B core protein or ubiquitin, plants expressing said protein, and uses thereof as vaccine
WO2011043962A2 (en) 2009-10-07 2011-04-14 Wyeth Llc Compositions comprising adjuvant, macrolide and proteinaceous antigen and methods of use thereof
WO2011043962A3 (en) * 2009-10-07 2011-06-03 Wyeth Llc Compositions comprising adjuvant, macrolide and proteinaceous antigen and methods of use thereof
US20190145981A1 (en) * 2016-04-21 2019-05-16 Sphingotec Therapeutics Gmbh Methods for determining dpp3 and therapeutic methods

Also Published As

Publication number Publication date
HUT71209A (en) 1995-11-28
GB9312324D0 (en) 1993-07-28
NZ267139A (en) 1998-01-26
DK0703789T3 (da) 1999-09-20
EP0703789A1 (de) 1996-04-03
DE69416405D1 (de) 1999-03-18
ZA944241B (en) 1995-05-26
ATE176402T1 (de) 1999-02-15
AU694121B2 (en) 1998-07-16
US5885814A (en) 1999-03-23
HU9403591D0 (en) 1995-03-28
GR3030116T3 (en) 1999-07-30
ES2130426T3 (es) 1999-07-01
DE69416405T2 (de) 1999-09-09
JPH08511262A (ja) 1996-11-26
AU6933394A (en) 1995-01-03
EP0703789B1 (de) 1999-02-03

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